Single molecule fluorescence detection methods can provide insights into the stochastic dynamics and structural distribution of proteins. Here we propose to develop both biochemical and spectroscopic tools to facilitate the application of single molecule methods to the study of protein structure and function. These include: (i) methods for selective derivatization of proteins with appropriate linkers and dyes for fluorescence studies and surface immobilization; and (ii) instrumentation and data analysis tools/methods for single molecule fluorescence detection. These methods will be applied to three well-defined biochemical systems: (i) the enzymatic hydrolysis of peptides and proteins by trysin; (ii) the folding of apomyoglobin; and (iii) studies of membrane protein association and diffusion with C5a receptor.